Project 463234

ABC transporters are a large family of integral membrane proteins critical to proper cell function. They also play key roles in a variety of human diseases, including drug resistance in many cancers, and have become targets of clinical interest. Despite the importance of these proteins, however, their biochemical complexity has made them challenging to study. Consequently, there is still much to learn about their regulatory interactions, as well as how changes in these interactions lead to disease. To address the challenges of studying complex, integral membrane proteins at a large scale we recently developed Mammalian Membrane Two-Hybrid High Throughput Screening (MaMTH-HTS). Building upon our previously reported MaMTH assay, MaMTH-HTS allows for sensitive detection of protein-protein interaction (PPIs) involving full-length integral membrane proteins directly in the context of living mammalian cells, but at a scale far greater than that of our original assay. Using MaMTH-HTS we built a detailed map of the PPIs of the ABC transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), identifying candidate proteins with potential roles in its regulation and cystic fibrosis. Here we propose to expand the use of our MaMTH-HTS system, in conjunction with an established pipeline of functional validation assays, to build comprehensive interaction maps of the remaining 43 integral membrane human ABC transporters, providing a global overview of the complex protein networks involved in their function and regulation. Additionally, we plan to expand our functional exploration of several interesting candidate proteins identified in our successful mapping of CFTR. These results should serve as a valuable resource for researchers, opening new avenues of exploration to improve our understanding of how various ABC transporter PPIs contribute to cancer drug resistance and genetic disease, and facilitate identification of new targets for therapeutic intervention.